Abstract
Endocytosis in neuronal dendrites is known to play a critical role in synaptic transmission and plasticity such as long-term depression (LTD). However, the inability to detect endocytosis directly in living neurons has hampered studies of its dynamics and regulation. Here, we visualized the formation of individual endocytic vesicles containing pHluorin-tagged receptors with high temporal resolution in the dendrites of cultured hippocampal neurons. We show that transferrin receptors (TfRs) are constitutively internalized at optically static clathrin-coated structures. These structures are slightly enriched near synapses that represent preferential sites for the endocytosis of postsynaptic AMPA-type receptors (AMPARs), but not for non-synaptic TfRs. Moreover, the frequency of AMPAR endocytosis events increases after the induction of NMDAR-dependent chemical LTD, but the activity of perisynaptic endocytic zones is not differentially regulated. We conclude that endocytosis is a highly dynamic and stereotyped process that internalizes receptors in precisely localized endocytic zones.
Highlights
The pulsed pH (ppH) Assay Reveals the Activity of clathrincoated structures (CCSs) in Neurons We applied the ppH assay to hippocampal neurons in culture transfected with the transferrin receptor fused to superecliptic pHluorin (SEP) (TfRSEP), a receptor constitutively internalized through Clathrin-mediated endocytosis (CME)
transferrin receptors (TfRs)-SEP receptors visible at pH 7.4 were colocalized with clathrin light chain fused to mCherry (Clc-mCh), and most of the signal disappeared at pH 5.5, revealing intracellular structures (Figure 1A)
The appearance of a spot in an image taken during a pH-5.5 interval indicates the formation of a clathrin-coated vesicle (CCV) that pinched off from the plasma membrane during the preceding 2-s interval, when the extracellular medium was at pH 7.4 (Figure 1B)
Summary
Molecular and cellular mechanisms regulating postsynaptic AMPAR trafficking have been studied in great detail (Anggono and Huganir, 2012), but key questions remain unanswered, such as where and when receptors are being internalized following NMDAR stimulation This issue is relevant, considering the synaptic specificity of LTD. Ultrastructural studies seldom identify clathrin-coated invaginations within spine heads but, rather, at the base of spine necks or in dendritic shafts (Cooney et al, 2002; Tao-Cheng et al, 2011) To distinguish between these two non-exclusive models, it would be necessary to detect the activity of endocytic zones and monitor their modulation during synaptic plasticity in living neurons
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